Interferon is a protein, which plays an important role in adaptive immune response against viruses.
Because of its important role in the adaptive immune system, there have been many studies on the role of the interferon in the immune system. However, it has been only limited knowledge on a signaling pathway for in-vivo production of interferon in the immune system responds to pathogens.
After recent discovery of Toll-like receptor (hereinafter just referred to as “TLR” for abbreviation where appropriate) as a receptor that recognize pathogens in mammal innate immune system, researches on TLR are leading to the understanding of the signaling pathway relating to the pathogen recognition in the innate immune system.
The mammalian TLR families, which are principal tissues appearing to be in large part conserved across Drosophila and human, recognize a variety of microbial components and mediate (i) activation of nuclear factor κB (hereinafter just referred to as “NF-κB” for abbreviation) and (ii) other signaling pathways.
For human, 10 receptors (human TLR 1 to 10) belonging to the TLR family have been identified by now and mouse homologue thereof (mouse TLR 1 to 10) have been identified. TLR family proteins is made of (i) an extracellular domain containing a plurality of leucine-rich repeats (LRRs) and a carboxylic terminal (C-terminal) flanking region, and (ii) a cytoplasmic (intercellular) signaling domain. The cytoplasmic signaling domain is called Toll/interleucine-1 receptor homology domain (TIR). Each TLR recognizes one or more distinct ligand(s) with its extracellular domain, and induces immune response(s), presumably via the intercellular TIR. Each TLR induces different, sometimes overlapping immune responses. All TLR family proteins contain a TIR domain in their cytoplasmic region, and most of the TIR domain is considered to be responsible for signaling and interaction with the adaptor molecule MyD88 or Mal/TIRAP. That is, TLR2, TLR4, TLR5, TLR7, and TLR9 transmit signals via the adaptor molecule MyD88 upon agonist stimulation, thereby to activate NF-κB.
Meanwhile, it was reported recently that the adaptor molecule Mal/TIRAP (an adaptor molecule called MyD88-adapter-like or TIRAP), which associates with TLR 4, relates to the signaling pathway via TLR 4, (e.g. refer to Document 1: Kawai, T., et a. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167: 5887-5894 (2001), Document 2: Horng, T., Barton, G. M., & Medzhitov, R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat. Immunol. 2: 835-841 (2001), and Document 3: Fitzgerald, K. A., et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 413: 78-83 (2001)).
According to the report, TLR 4 is involved in activation of NF-κB, MAPK, and interferon β promoter. This unique ability of TLR 4 to induce the activation of interferon β promoter is ascribed to signaling pathway mediated by the adaptor molecule Mal/TIRAP that binds to TLR 4. This signaling pathway is called “a MyD88-independent pathway”. That is, in the signaling via TLR 4, activation of NF-κB and type-I interferon promoter is controlled by cooperation between TLR 4 and the adaptor molecule Mal/TRRAP that is a second adaptor molecule different from the adaptor molecule MyD88.
In Microphages (Mf), STAT1 α/β phosphorylation is induced by the interferon β activation as a result of TLR 4 stimulation, not TLR 2 stimulation. Expression of the gene encoding interferon β subsequently induces production of MCP (Monocyte Chemoattractant Protein)-5, IP (interferon Inductive Protein)-10 m, and iNOS (inductive NO synthetic enzyme). Again, this occurs via the MyD88-independent pathway, even in MyD88−/−cell (a cell from which the adaptor molecule MyD88 is deleted).
A current concept considered most likely is that the adaptor molecule Mal/TIRAP covers the MyD88-independent pathway.
In contrast, the inventor of the present invention studied immune responses induced by double-stranded RNA and mediated via human TLR 3, and found that human TLR 3 relates to recognition of double-stranded RNA on a cell surface of human fibroblast, and triggers downstream signaling that induces the interferon β production. (e.g. Document 4: Matsumoto, M., Kikkawa, S., Kohase, M., Miyake, K., & Seya, T. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem. Biophys. Res. Commun. 293: 1364-1369 (electrically published on May 31, 2002). That is, the inventors of the present invention showed that the interferon β promoter activation and interferon β production is rapidly and strongly induced by human TLR 3 in response to double-stranded RNA. Reporter gene assay showed that human TLR3 mediates the interferon β promoter activation, and to a lesser extent the NF-κB activation (c.f. Document 4 for example). This result with respect to TLR3 is quite different from those of TLR, TLR2, TLR5, TLR7, and TL9, which activate NF-κB and p38 MAPK (MAP kinase) through the adaptor molecule MyD88 after recognizing their specific ligands.
The type I interferon (interferon a and interferon β) that TLR 3 induces is known to have anti-virus effect and anti-cancer effect. Specifically, type I interferon has the following effects.
Type I interferon is known to exhibit the anti-virus effect by the following function mechanisms:
1) The type I interferon destabilizes mRNA of a virus and activates intercellular gene that inhibits protein translation of hosts, thereby to inhibit replication and multiplication of the virus;
2) The type I interferon induces expression of MHC class I molecule so as to induce resistance against natural killer (NK) cells. Further, type I interferon enhances sensitivity with respect to CD+8 cytotoxic T cell. In addition, type I interferon participates to inhibition of T cell activation and to T cell suppressor activation; and
3) The type I interferon activates natural killer (NK) cells that selectively damage virus-infected cells, and causes NK-cell induced apoptosis in the virus. Moreover, the type I interferon is known to exhibit anti-tumor effect by the following function mechanisms:
1) The type I interferon destabilizes mRNA in tumor cells and activates intercellular gene that inhibits protein translation of hosts. This inhibits protein synthesis in the tumor cells, thereby inhibiting multiplication of the tumor cells;
2) The type I interferon activates anti-tumor effectors such as microphages, NK cells, natural killer T (NKT) cells, and the like. Via damaging of the tumor cells by these anti-tumor effectors, apoptosis is brought about in the tumor cells;
3) The type I interferon activates NK cells that selectively damage virus-infected cells, and induces NK cell-induced apoptosis in the tumor cells.
Further, as described above, the type I interferon participates inhibition of T cell activation, and enhancement of T cell suppressor activity. Therefore, it is considered that some kinds of autoimmune diseases can be ameliorated by the type I interferon.
Because of the anti-virus effect and anti-tumor effect as mentioned above of the type I interferon, interferon α formulation and interferon β formulation have been used for treating hepatitis B, hepatitis C, hepatitis C-induced liver and kidney cancers, and the like. For example, “Sumiferon (Registered Trademark)” made by Sumitomo Pharmaceuticals, which is a wild type interferon a, are used successfully in clinical applications.
However, the researches on TLR that have been carried out so far indicate that the signaling pathway leading to the type I interferon production induced by TLR3 witch recognized double-stranded RNA is different from the signaling pathways mediated via other TLRs. However, it has not been understood which protein participates in the signaling pathway.
It is believed that discovery of existence of a protein that induces the signaling by specifically binding to TLR3, the signaling causing the type I interferon production in downstream will lead to understanding of a signaling pathway important for innate immune response against viruses and a control mechanism of the signaling pathway. The understanding of the signaling pathway and its control mechanism is expected to be used in pathological analysis of various illnesses relating to the innate immune system and development of therapeutic agents that control innate immune response.
Moreover, the interferon formulations used for viral infectious diseases and tumors is for systemic administration: at therapeutic concentrations the interferon formulations have strong side effects such as worsening pschoneurosis (depression and the like), autoimmune disease (thyroid insufficiency, autoimmune hepatitis, hemolytic anemia, ulcerative colitis, rheumatoid arthritis, and the like), and the like side effect. The side effects caused by the interferon formulations occur presumably because interferon is introduced even in normal host cells that do not produce interferon, thereby inducing presentation of autoantigen to T cells in all portions where the interferon is induced, and thus making it easy to cause autoimmune phenomenon to occur, even though the presentation of autoantigen to T cells is normally caused when immune responds to exogenous antigen.
Because they have strong side effects at their therapeutic concentration, the interferon formulations have difficulties to maintain sufficient anti-cancer effect if systemically administered. Moreover, even if administered locally, it is considered that the side effects are difficult to avoid completely.
It is considered that if a novel protein that specifically binds to TLR3 so as to induce the downstream signaling that leads to the type I interferon production is found out, it will be possible to develop a new therapeutic agent for treating a viral infectious disease, tumor, or the like by locally enhancing the type I interferon production in vivo.
Moreover, functional analysis of the novel protein is expected to provide a protein that inhibits the type I interferon production in vivo. Further it will be possible to provide a therapeutic agent that inhibits the type I interferon production in vivo by this protein thereby treating autoimmune disease, atopic disease, and the like.
In view of the foregoing problems, an object of the present invention is to provide a novel adaptor protein and its mutants, a gene of the protein, a recombinant expression vector including the gene, an antigen against the protein, and a therapeutic agent for preventing or treating a viral infectious disease, a therapeutic agent for treating a tumor, a therapeutic agent for treating autoimmune disease and atopic disease.